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抗牛PEPCK蛋白单克隆抗体的制备和初步鉴定.docx

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抗牛PEPCK蛋白单克隆抗体的制备和初步鉴定 摘要：本文主要介绍抗牛PEPCK蛋白单克隆抗体（mAb）的制备和初步鉴定。利用大肠杆菌表达系统表达牛PEPCK蛋白并纯化，通过动物免疫和细胞融合技术制备出一株具有高亲和力和特异性的mAb，并对其进行初步鉴定，包括免疫组化、间接ELISA和免疫印迹等方法。结果表明，所制备的mAb可特异性识别牛PEPCK蛋白。 关键词：抗体；克隆；PEPCK蛋白；特异性识别 Introduction Phosphoenolpyruvatecarboxykinase(PEPCK)isoneoftheenzymesinvolvedintheprocessofgluconeogenesis,whichplaysavitalroleinmaintainingglucosehomeostasisinanimals.PEPCKiswidelyexpressedinmanytissues,especiallyinliver,kidney,andadiposetissue.Therefore,itisimportanttodevelopaspecificandsensitivemethodtodetectPEPCKproteinindifferentphysiologicalandpathologicalconditions.Themonoclonalantibody(mAb)isapowerfultoolforproteindetection,andithashighspecificityandsensitivity.Inthisstudy,weaimedtoprepareaspecificmAbagainstbovinePEPCKprotein. MaterialsandMethods PreparationofbovinePEPCKprotein ThecodingsequenceofbovinePEPCKwasclonedintotheexpressionvectorpET-28a(+)withaHis-tagattheN-terminal.TherecombinantplasmidwastransformedintotheBL21(DE3)strainofEscherichiacoli.Thecellswereculturedat37°CuntiltheA600reached0.6andinducedwith0.2mMIPTGat16°Cfor16h.Thecellswereharvestedbycentrifugationandlysedbysonication.TheHis-taggedPEPCKproteinwaspurifiedbyNi-NTAchromatography. Animalimmunizationandhybridomageneration FemaleBalb/cmice(6-8weeksold)wereimmunizedwithpurifiedbovinePEPCKproteinmixedwithFreund'scompleteadjuvantforthefirstimmunizationandFreund'sincompleteadjuvantforsubsequentimmunizations.Afterfourimmunizations,thespleencellsfromimmunizedmicewerefusedwithmyelomaSP2/0cellsusingpolyethyleneglycol(PEG).ThehybridswereselectedinHAT(hypoxanthine-aminopterin-thymidine)medium.Theclonesproducinganti-PEPCKantibodywerescreenedbyindirectELISA. ProductionandpurificationofmAb Theselectedpositivehybridomacellswereculturedinserum-freemedium,andtheculturesupernatantswerecollectedandpurifiedusingproteinA-Sepharose.ThepurifiedmAbwasdialyzedagainstPBSandsterilefiltered. CharacterizationofthemAb ThespecificityofmAbwasevaluatedbyimmunohistochemistry,indirectELISA,andWesternblotting.Forimmunohistochemis